Histamine is a biogenic amine that is indispensable in the efficient functioning of various physiological systems. In previous work, a molecularly imprinted polymer (MIP) based sensor platform with impedimetric read-out was presented which could rapidly and at low cost determine histamine concentrations in buffer solutions within pH 7-9. For diagnostic applications, histamine should be detectable in a wider pH range as it mostly occurs in mildly acidic environments. To understand this pH-dependent response of the MIP sensor, we propose a statistical binding analysis model. Within this model, we predict the theoretical performance of MIP based on acrylic acid in the required pH range and verify these results experimentally by UV-vis spectroscopy, microgravimetry, and impedance spectroscopy. Using impedimetric read-out, specific and selective detection of histamine in the physiologically relevant nanomolar concentration range is possible in neutral and mildly acidic phosphate buffer. Finally, this sensor platform was used to analyze the histamine concentration of mildly acidic bowel fluid samples of several test persons. We show that this sensor provides reliable data in the relevant concentration regime, which was validated independently by enzyme-linked immuno sorbent assay (ELISA) tests.

Surface bioconjugation of biomolecules has gained enormous attention for developing advanced biomaterials including biosensors. While conventional immobilization (by physisorption or covalent couplings using the functional groups of the endogenous amino acids) usually results in surfaces with low activity, reproducibility and reusability, the application of methods that allow for a covalent and uniformly oriented coupling can circumvent these limitations. In this study, the nanobody targeting Vascular Cell Adhesion Molecule-1 (NbVCAM1), an atherosclerotic biomarker, is engineered with a C-terminal alkyne function via Expressed Protein Ligation (EPL). Conjugation of this nanobody to azidified silicon wafers and Biacore™ C1 sensor chips is achieved via Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) “click” chemistry to detect VCAM1 binding via ellipsometry and surface plasmon resonance (SPR), respectively. The resulting surfaces, covered with uniformly oriented nanobodies, clearly show an increased antigen binding affinity, sensitivity, detection limit, quantitation limit and reusability as compared to surfaces prepared by random conjugation. These findings demonstrate the added value of a combined EPL and CuAAC approach as it results in strong control over the surface orientation of the nanobodies and an improved detecting power of their targets—a must for the development of advanced miniaturized, multi-biomarker biosensor platforms.

With the current trend to miniaturize bioactive surfaces to micro-or nanometer scale, the strategy of immobilization becomes more important. Therefore, there is a growing need for protein immobilization techniques that create both stable and homogeneously covered surfaces in a reproducible way. One of the most promising methods to achieve this is the combination of biological receptors with 'click' chemistry, like the Copper catalyzed Alkyne Azide Cycloaddition (CuAAC). This work presents a full optimization of all aspects of the 'click' chemistry reaction between proteins and surfaces in order to create covalently and homogeneously covered biosurfaces. The coupling procedure is monitored by in situ ellipsometry, a unique characterization technique that offers the opportunity to quantify minute amounts of surface coupled protein mass in real-time. The optimization involves the azidification of a solid silicon support, the alkynation of two proteins, Staphylococcus aureus Protein A (SpA) and Maltose Binding Protein (MBP), as well as the development of a highly reproducible CuAAC 'click' coupling protocol. Using the here optimized protocol, active and reproducible biolayers can be created rapidly. The proposed surface biofunctionalization method combined with ellipsometry forms a unique and promising platform towards the development of highly sensitive, accurate biosensors.